Mycobacterium tuberculosis accounts for over 2 million deaths per year. Furthermore, the global burden of tuberculosis has been compounded by its deadly association with the AIDS virus and by the emergence of multi-drug resistant strains, which have increased the demand for new treatments to stem the tuberculosis/AIDS epidemic. The design of new drugs and vaccines requires an understanding of the biology of mycobacteria and the development of genetic tools to manipulate their genomes in order to determine the molecular basis of pathogenesis and drug resistance. Although both transformation and transduction have had important roles in the development of mycobacterial genetics, little is known about conjugal DNA transfer. Conjugation offers an important alternative for transferring DNA between mycobacteria and, in particular, as a gene delivery system for moving markers between strains and generating targeted mutations. During the last grant-period, we described a DNA transfer process in Mycobacterium smegmatis that is different from any conjugation system described to-date. This proposal is designed to characterize the M. smegmatis conjugation system by defining and identifying both cis-acting DNA sequences and trans-acting proteins that mediate DNA transfer. Such analyses will provide important mechanistic information about the process of DNA transfer and allow differences between donor and recipient cells to be determined. Moreover, by modifying transferable plasmids, a new allele-exchange system will be established, enabling the capture of segments of chromosomal DNA and the generation of targeted mutations by transfer-mediated recombination. The application of this system to the slow-growing mycobacterial pathogens will be a valuable new addition to current molecular approaches. In addition, plasmids have been isolated from Mycobacterium avium that encode DNA relaxases related to those required for classical conjugal transfer in gram-negative bacteria. The ability of these plasmids to transfer among slow-growing mycobacteria will be investigated to understand the role of conjugation in lateral transfer among mycobacterial populations and its possible role in the spread of drug resistance. The aims are: 1. To characterize cis-acting sequences required for DNA transfer and to develop transfer as a molecular genetic tool for the study of mycobacteria. 2. To identify and characterize trans-acting transfer functions in both donor and recipient cells. 3. To examine transfer of chromosomal and plasmid DNA among fast- and slow-growing mycobacteria.